The invention relates generally to novel nucleomonomer and oligomer analogs, and to oligonucleotide-based therapy and diagnosis by binding of the oligonucleotide analogs to single or double-stranded nucleic acid target sequences. More specifically, the invention concerns oligomers containing certain 5-substituted pyrimidine base residues and intermediates in their synthesis.
Sequence-specific binding of oligonucleotides both to single-stranded RNA and DNA and to duplex DNA has been demonstrated. The appropriate sequence recognition for binding to single-stranded targets is well known: the A-T and G-C pairing characteristic of duplex formation has been established as the basis for DNA replication and transcription.
More recently, oligonucleotides have been shown to bind in a sequence-specific manner to duplex DNA to form triplexes. Single-stranded nucleic acid, primarily RNA, is the target molecule for oligonucleotides that are used to inhibit gene expression by an xe2x80x9cantisensexe2x80x9d mechanism (Uhlmann, E., et al, Chem Reviews (1990) 90:543-584; van der Krol, A. R., et al, Biotechniques (1988) 6:958-976). Antisense oligonucleotides are postulated to exert an effect on target gene expression by hybridizing with a complementary RNA sequence. In this model, the hybrid RNA-oligonucleotide duplex interferes with one or more aspects of RNA metabolism including processing, translation and metabolic turnover. Chemically modified oligonucleotides have been used to enhance their nuclease stability.
Duplex DNA can be specifically recognized by oligomers based on a recognizable nucleomonomer sequence. Two major recognition motifs have been recognized. In an earlier description of a xe2x80x9cCTxe2x80x9d motif, protonated cytosine residues recognize G-C basepairs while thymine residues recognize A-T basepairs in the duplex. These recognition rules are outlined by Maher III, L. J., et al., Science (1989) 245:725-730; Moser, H. E., et al., Science (1987) 238:645-650. More recently, an additional motif, termed xe2x80x9cGTxe2x80x9d recognition, has been described (Beal, P. A., et al, Science (1992) 25:1360-1363; Cooney, M., et al., Science (1988) 241:456-459; Hogan, M. E., et al., EP Publication 375408). In the G-T motif, A-T pairs are recognized by adenine or thymine residues and G-C pairs by guanine residues.
In both of these binding motifs, the recognition sequence of the oligomer must align with the complementary sequence of the purine chain of the duplex; thus, recognition, for example, of an A-T pair by a thymine, depends on the location of the adenyl residues along the purine chain of the duplex. An exception to the foregoing is the recent report by Griffin, L. C., et al., Science (1989) 245:967-971, that limited numbers of guanine residues can be provided within pyrimidine-rich oligomers and specifically recognize thymine-adenine base pairs; this permits the inclusion of at least a limited number of pyrimidine residues in the homopurine target.
The two motifs exhibit opposite binding orientations with regard to homopurine target chains in the duplex. In the CT motif, the targeting oligonucleotide is oriented parallel to the target purine-rich sequence; in the GT motif, the oligonucleotide is oriented antiparallel (Beal, P. A., et al., Science (1990) 25:1360-1363).
The efficiency of binding by C residues in CT motif oligomers is reduced as the pH of hybridization is increased. The protonated tautomer of C (C+) is the binding competent species in Hoogsteen binding, but is present at only low levels at physiological pH. This is consonant with the pKa of cytosine which is 4.25. Base analogs such as 5-methylcytosine, pKa 4.35, (Lee, J. S. et al., Nucleic Acids Res (1984) 12:6603-6614), 8-oxo-N6-methyladenine (Krawczyk, S. H. et al, Proc Natl Acad Sci (1992) 89:3761-3764; International Application No. PCT/US91/08811), pseudoisocytidine (Ono, A., et al, J Org Chem (1992) 57:3225-3230; International Application No. PCT/US90/03275) or carbocyclic cytidine (Froehler, B. C., et al, J Am Chem Soc (1992) 114:8320-8322; U.S. patent application Ser. No. 07/864,873 incorporated herein by reference) have been utilized to obtain binding of CT motif oligomers over an extended pH range.
Sequence-specific targeting of both single-stranded and duplex target sequences has applications in diagnosis, analysis, and therapy. Under some circumstances wherein such binding is to be effected, it is advantageous to stabilize the resulting duplex or triplex over long time periods.
Covalent crosslinking of the oligomer to the target provides one approach to prolong stabilization. Sequence-specific recognition of single-stranded DNA accompanied by covalent crosslinking has been reported by several groups. For example, Vlassov, V. V., et al., Nucleic Acids Res (1986) 14:4065-4076, describe covalent bonding of a single-stranded DNA fragment with alkylating derivatives of nucleomonomers complementary to target sequences. A report of similar work by the same group is that by Knorre, D. G., et al., Biochimie (1985) 67:785-789. Iverson and Dervan also showed sequence-specific cleavage of single-stranded DNA mediated by incorporation of a modified nucleomonomer which was capable of activating cleavage (J Am Chem Soc (1987) 109:1241-1243). Meyer, R. B., et al., J Am Chem Soc (1989) 111:8517-8519, effect covalent crosslinking to a target nucleomonomer using an alkylating agent complementary to the single-stranded target nucleomonomer sequence. Photoactivated crosslinking to single-stranded oligonucleotides mediated by psoralen was disclosed by Lee, B. L., et al., Biochemistry (1988) 27:3197-3203. Use of crosslinking in triple-helix forming probes was also disclosed by Horne, et al., J Am Chem Soc (1990) 112:2435-2437.
Use of N4,N4-ethanocytosine as an alkylating agent to crosslink to single-stranded and double-stranded oligomers has also been described (Webb and Matteucci, J Am Chem Soc (1986) 108:2764-2765; Nucleic Acids Res (1986) 14:7661-7674; Shaw, J. P., et al, J Am Chem Soc (1991) 13:7765-7766). These papers also describe the synthesis of oligonucleotides containing the derivatized cytosine. Matteucci and Webb, in a later article in Tetrahedron Letters (1987) 28:2469-2472, describe the synthesis of oligomers containing N6,N6-ethanoadenine and the crosslinking properties of this residue in the context of an oligonucleotide binding to a single-stranded DNA.
In a recent paper, Praseuth, D., et al., Proc Natl Acad Sci (USA) (1988) 85:1349-1353, described sequence-specific binding of an octathymidylate conjugated to a photoactivatable crosslinking agent to both single-stranded and double-stranded DNA.
In addition, Vlassov, V. V. et al., Gene (1988) 313-322 and Fedorova, O. S. et al., FEBS (1988) 228:273-276, describe targeting duplex DNA with an alkylating agent linked through a 5xe2x80x2-phosphate of an oligonucleotide.
In effecting binding to obtain a triplex, to provide for instances wherein purine residues are concentrated on one chain of the target and then on the opposite chain, oligomers of inverted polarity can be provided. By xe2x80x9cinverted polarityxe2x80x9d is meant that the oligomer contains tandem sequences which have opposite polarity, i. e., one having polarity 5xe2x80x2xe2x86x923xe2x80x2 followed by another with polarity 3xe2x80x2xe2x86x925xe2x80x2, or vice versa. This implies that these sequences are joined by linkages which can be thought of as effectively a 3xe2x80x2-3xe2x80x2 internucleoside junction (however the linkage is accomplished), or effectively a 5xe2x80x2-5xe2x80x2 internucleoside junction. Such oligomers have been suggested as by-products of reactions to obtain cyclic oligonucleotides by Capobionco, M. L., et al., Nucleic Acids Res (1990) 18:2661-2669. Compositions of xe2x80x9cparallel-stranded DNAxe2x80x9d designed to form hairpins secured with AT linkages using either a 3xe2x80x2-3xe2x80x2 inversion or a 5xe2x80x2-5xe2x80x2 inversion have been synthesized by van de Sande, J. H., et al., Science (1988) 241:551-557. In addition, triple helix formation using oligomers which contain 3xe2x80x2-3xe2x80x2 linkages have been described (Horne, D. A., and Dervan, P. B., J Am Chem Soc (1990) 112:2435-2437; Froehler, B. C., et al, Biochemistry (1992) 31:1603-1609).
The use of triple helix (or triplex) complexes as a means for inhibition of the expression of target gene expression has been previously adduced (International Application No. PCT/US89/05769). Triple helix structures have been shown to interfere with target gene expression (International Application No. PCT/US91/09321; Young, S. L. et al, Proc Natl Acad Sci (1991) 88:10023-10026), demonstrating the feasibility of this approach.
European Patent Application No. 92103712.3, Rahim, S. G., et al (Antiviral Chem Chemother (1992) 3:293-297), and International Application No. PCT/SE91/00653 describe pyrimidine nucleomonomer characterized by the presence of an unsaturated group in the 5-position. Propynyl and ethynyl groups are included among the derivatives at the 5-position that are described in the applications.
Synthesis of nucleomonomers having unsaturated alkyl groups at the 5-position of uracil has been described (DeClercq, E., et al, J Med Chem (1983) 26:661-666; Goodchild, J., et al, J Med Chem (1983)26:1252-1257). Oligomers containing 5-propynyl modified pyrimidines have been described (Froehler, B. C., et al, Tetrahedron Letters (1992) 33:5307-5310).
Conversion of 5-propynyl-2xe2x80x2-deoxyuridine, 5-butynyl-2xe2x80x2-deoxyuridine and related compounds to the 5xe2x80x2-triphosphate followed by incorporation of the monomer into oligomers by E. coli polymerase has been described (Valko, K., et al, J Liquid Chromatog (1989) 12:2103-2116; Valko, K. et al, J Chromatog (1990) 506:35-44). These studies were conducted as a structure to activity analysis of nucleotide analogs having a series of substitutions at the 5- position of uracil. The activity of the nucleotide analogs as substrates for E. coli polymerase was examined and correlated with characteristics such as the hydrophobicity of the monomer. No information was presented regarding the properties of oligomers containing the analogs.
European patent application 0492570 published Jul. 1, 1992 describes a method for detecting a target polynucleotide using a single-stranded polynucleotide probe in which an intercalating molecule is attached by a linker which comprises at least 3 carbon atoms and a double bond at the alpha position relative to the base.
PCT patent publication WO 92/02258 describes nuclease resistant, pyrimidine modified oligomers including a substituent-group at the 5 or 6 positions, including phenyl. 5-Phenyl-2xe2x80x2-deoxyuridine has been subsequently incorporated into oligomers and shown to decrease the binding affinity of oligomers containing this modification for both single stranded and double stranded target sequences.
DNA synthesis via amidite and hydrogen phosphonate chemistries has been described (U.S. Pat. Nos. 4,725,677; 4,415,732; 4,458,066; 4,959,463).
Oligomers having enhanced affinity for complementary target nucleic acid sequences would have improved properties for diagnostic applications, therapeutic applications and research reagents. Thus, a need exists for oligomers with enhanced binding affinity for complementary sequences. Oligomers of the present invention have improved binding affinity for double stranded and/or single stranded target sequences.